Process and equipment for plasmid purification

ABSTRACT

A scalable alkaline lysis process, including procedures and devices for the isolation of large quantities (grams and kilograms) of plasmid DNA from recombinant  E. coli  cells. Effective, controllable, and economical operation, and consistent low level of host chromosomal DNA in the final plasmid product. Involves a series of new unit operations and devices for cell resuspension, cell lysis, and neutralization.

RELATED APPLICATIONS

[0001] This application claims priority to co-pending U.S. patentapplication Ser. No. 08/887,673, filed Jul. 3, 1997, which in turn,claims priority to U.S. Provisional Patent Application Ser. No.60/022,157, filed Jul. 19, 1996. Both applications are herebyincorporated by reference as if fully set forth herein.

FIELD OF INVENTION

[0002] The present invention relates to the field of plasmidpurification and is particularly useful in the field of gene therapy.

BACKGROUND OF THE INVENTION

[0003] The following description of the background of the invention isprovided to aid in understanding the claimed invention, but it is notadmitted to constitute or describe prior art to the claimed inventionand should in no way be construed as limiting the claimed invention.

[0004] The traditional alkaline lysis process for isolation of plasmidDNA from bacterial cells is described in Birnboim, H. C. and J. Doly,Nucleic Acids Res. 7:1513, 1979 and is also described in J. F. Burke,Nucleic Acids Res. 8:2989, 1981. It makes use of severallaboratory-scale apparatuses and manual operations which are notsuitable for large-scale manufacturing. In addition, the traditionalalkaline lysis process is not suitable for making pharmaceutical gradematerial for human use, since inconsistent process performance mayresult in an unacceptable level of sheared host chromosomal DNA in thefinal plasmid product. The difficulty of theoretical study of some ofthe critical parameters for manual operations, like shear force, alsomake the traditional process unlikely to become a large scalemanufacturing process.

[0005] Such traditional processes consist of three stages. The firststage is cell resuspension, which normally utilizes manual stirring or amagnetic stirrer, and a homogenizer or impeller mixer to resuspend cellsin the resuspension buffer. Manual stirring and magnetic stirring areonly appropriate for laboratory scale preparations.

[0006] The second stage is cell lysis. It is desirable to minimize shearof host chromosomal DNA at this stage, because it is difficult to removesheared chromosomal DNA in the downstream purification process (due tothe similarity in properties between sheared chromosomal DNA and plasmidDNA). The lysis is normally carried out by manual swirling or magneticstirring in order to mix the resuspended cells with lysis solution(consisting of diluted alkali (base) and detergents); then holding themixture at room temperature (20-25 degrees Celsius) or on ice for aperiod of time, such as 5 minutes, to complete lysis. As noted above,manual swirling and magnetic stirring are not scalable. In addition, itis difficult to optimize the process and obtain consistent processperformance due to the number of operation parameters, includingoperator to operator variability.

[0007] The third stage is neutralization and precipitation of hostcontaminants. Lysate from the second stage is normally mixed with a coldneutralization solution by gentle swirling or magnetic stirring toacidify the lysate before setting in ice for 10-30 minutes to facilitatethe denaturation and precipitation of high molecular weight chromosomalDNA, host proteins, and other host molecules. Again, both manualswirling and magnetic, stirring are not scalable. An ice bath is notconvenient if a long holding time is desired in a large scale process,because a large quantity of water must be removed and a large quantityof ice/or ice-NaCl mixture needs to be added periodically to maintain asteady temperature.

[0008] Once the plasmid DNA is extracted from the lysed cells itspurification has become a routine and important procedure for themolecular biologist. However, the scale for these purifications, oftenreferred to as “mini-preps”, is usually less than about 1 milligram ofplasmid DNA. These small scale preps isolate plasmid DNA from thesupernatant of lysed bacterial cells using a variety of techniques, suchas ethanol precipitation. For slightly larger scale preparations, theprimary techniques employed use cesium chloride centrifugation, bindingand eluting to silica resins (in the presence of chaotropic salts) orbinding and eluting with various anionic chromatography resins. Inaddition, other techniques are sometimes used in combination with theresins mentioned, e.g., PEG and/or alcohol precipitation, RNasetreatment, and phenol/chloroform extraction. There are also some plasmidpurifications performed using analytical HPLC, in particular reversephase HPLC to separate different plasmid forms using organic solventsystems.

[0009] Reverse phase chromatography (“RPC”) is generally practiced bybinding compounds of interest to a chromatography support in an aqueoussolution and eluting with increasing amounts of an organic solvent, suchas acetonitrile or alcohol. This approach has been used by a number ofinvestigators to separate DNA, especially oligonucleotides and small(less than 1,000 base pair) restriction fragments. It has also been usedto separate open circular and supercoiled plasmids. However, RPCsolvents present volatility, fire, health and waste disposal risks. Inaddition, RPC of nucleic acids frequently requires the use of anion-pairing agent (e.g., triethylammonium acetate or TEAA) which can bedifficult to remove from the DNA and can be toxic to cultured cells.

[0010] The “mini-prep” procedures described above were designed forpurifying small amounts of plasmid DNA and in general they have not beensuitable for large-scale, high throughput purification processes. Largescale purification of plasmid DNA may magnify the contaminants in thefinal purifications in the final preparations, which usually goundetected in mini-preps. Anion-exchange chromatography as a singlechromatography step is unlikely to remove enough of the contaminantswhich would be necessary for a therapeutic product. Thus, additionalprocesses should probably be included to increase the purity of theplasmid DNA. In addition, it would be useful to have a scalable processwhich could also resolve various plasmid forms.

[0011] Two contaminants which may be particularly troublesome are RNAand chromosomal DNA. Many mini-prep procedures attempt to remove RNAusing one or several RNase enzymes which degrade the RNA toribonucleotides and small oligoribonucleotides. These can then beseparated from the plasmid DNA using any of a variety of techniques,including alcohol precipitation, size exclusion chromatography, anionexchange chromatography, etc. However, the use of RNase is undesirablein large scale (equal to or greater than 50-100 mg of plasmid)purification. RNase is an expensive material that is generally notreused. Large scale RNase reactions can be difficult to perform in batchmode with appropriate control of time, temperature and other reactionconditions. Also, RNase is typically isolated from bovine pancreas. Assuch, it is a possible source of mammalian pathogens, especiallyretrovirus and bovine spongiform encephalopathy (BSE). Use of suchmaterials in making plasmids for human use presents significant safetyand regulatory issues.

[0012] Another approach to removal of RNA involves differentialprecipitations whereby plasmid DNA is precipitated while RNA remains insolution or vice versa. An example is described in WO 95/21250 in whichpolyethylene glycol (PEG) is used to precipitate RNA from a solutioncontaining both RNA and plasmid. Similar techniques have been described.A disadvantage of these techniques as typically practiced is that theplasmid is first partially purified from the lysate by removing soliddebris, precipitated proteins and other solids, and optionally byalcohol precipitation of the nucleic acids (including RNA and plasmid).Differential precipitation is then applied to the partially purifiedmixture of RNA and plasmid. This approach to differential precipitationhas required multiple steps, increasing the time effort and complexityof the process, and introducing more opportunities for loss of plasmid.

[0013] Removing chromosomal DNA derived from the bacterial host is alsoa challenging task in plasmid purification. In a typical “mini-prep”,chromosomal DNA is removed primarily during the lysis and neutralizationsteps. Large fragments of chromosomal DNA are bound to proteins andmembrane fragments, and are carried into the precipitate during theneutralization step. However, it is well known that if the chromosomalDNA is sheared to smaller size (≦ about 10 kb), it is not efficientlyprecipitated and contaminates the plasmid DNA. Thus, it has been assumedthat during mini preps, one must avoid vigorous mixing and shearing ofthe lysate. This is difficult to achieve at larger scales due to theknown difficulties of mixing large volumes of liquid. Thus, it isdesirable to have a way to separate plasmid DNA from chromosomal DNAsubsequent to lysis and neutralization.

[0014] One approach to this is CsCl/ethidium bromide density gradients.These are very effective for small amounts of plasmid (≦ 1 mg) that arenot intended for human use. They are not generally suitable for scalingup to over 100 mg lots due to the high cost of necessary equipment. Theyare also not generally suitable for producing plasmid for human usebecause ethidium bromide binds tightly to DNA, is difficult to removequantitatively, and is a known mutagen and suspected carcinogen.

[0015] Thus, there are several clear needs for large scale plasmidpurification. These include a method to mix large volumes of lysate withlow shear, a method to precipitate RNA directly from a lysate withoutprior additional purification, and a method to separate shearedchromosomal DNA from plasmid DNA.

[0016] Other methods for purification of larger amounts of plasmid DNAare not ideal and leave significant room for improvement. For example,the method described in WO 95/21250, published August 10, 1995 involvesmultiple precipitations, plasmid precipitations, low capacity sizeexclusions, and requires flammable alcohols. Similarly, the methoddescribed in WO 96/02658, published Feb. 1, 1996, requires lysozyme, mayrequire RNase, and requires flammable alcohols.

SUMMARY OF THE INVENTION

[0017] The present invention relates to a scalable alkaline lysisprocess, including procedures and devices for the isolation of largequantities (grams and kilograms) of plasmid DNA from recombinant E. colicells. The present invention provides effective, controllable, andeconomical operation, and consistent low level of host chromosomal DNAin the final plasmid product. These attributes, and the details whichfollow, clearly provide advantages over the traditional alkaline lysisprocess and the methods proposed by others for commercial scaleprocedures.

[0018] The present invention exploits a set of devices and procedurescapable of providing fast and efficient cell resuspension in asemi-continuous mode. All device's design and operation parameters canbe well characterized and optimized through empirical tests in bothscale-down and scale-up processes. Both mixing quality and shear forcecan be well controlled to maximize mixing efficiency and to minimizepossible damage to cells.

[0019] The present invention also provides a set of devices andprocedures capable of providing efficient and gentle mixing and celllysis in a continuous flow mode. The device's design (e.g. devicedimension) and operation parameters (e.g. flow rate and residence time)were well characterized through empirical tests and theoreticalcalculations and modeling to maximize lysis efficiency and minimizeshearing of chromosomal DNA. Surprisingly, by performing alkaline lysisusing a high concentration of unbuffered salt, not only is denaturedchromosomal DNA, protein, and cellular debris trapped in a precipitablesalt/detergent complex, but a significant portion of RNA is alsoprecipitated, thereby eliminating the need for an RNase treatment stepand providing a significant advantage.

[0020] In addition, the present invention provides a set of devices andprocedures capable of providing fast chilling and efficient and gentlemixing to denature and precipitate chromosomal DNA, protein, and RNA.The devices' selection and design and operation parameters were wellcharacterized through scale-up-empirical testing.

[0021] The invention relies, in part on the use of anion exchangechromatography, preferably with a Fractogel EMD 650S TMAE(S) resin witha 20-40 micron size. Other tentacle resins or means for making bindingsites more available to larger molecules like plasmids can also be used.The tentacles are preferably 15 to 50 units in length and have anaverage of 18 charged groups covalently bound to each tentacle.

[0022] The use of such resins: (1) provides a high plasmid DNA bindingcapacity (about 3 mg/mL, preferably about 1.5 mg/mL); (2) allows forefficient removal of proteins, RNA, low molecular weight molecules andprobably some chromosomal DNA and some open circle plasmid DNA; and (3)provides a means for enriching the supercoiled plasmid DNA above about80% using a step gradient. Supercoiled plasmid binds tighter to theFractogel resin (high affinity sites) allowing the remaining RNA andsome open circle plasmid to be removed.

[0023] The invention also relies in part on the use of hydrophobicinteraction chromatography, which is used to separate plasmid DNA fromE. coli chromosomal DNA and RNA and may also be used to separate opencircular plasmid DNA from supercoiled DNA. Overall, HIC is a powerfultechnique for plasmid DNA purification. This disclosure reveals thesurprising and unexpected value that hydrophobic interactionchromatography (HIC) has, especially when used in conjunction withanion-exchange chromatography, for large-scale plasmid DNA purification.Particularly surprising is the ability of HIC to resolve the supercoilform of a plasmid from the relaxed open circle form. Supercoiled DNA maybe easier to formulate and with certain formulations supercoiled plasmidmay have higher expression levels in vivo (e.g., about 10 times greaterexpression in certain systems). Another surprising and important fact isthat removal of chromosomal DNA, denatured plasmids, RNA, and endotoxinfrom the plasmid DNA forms can also be achieved.

[0024] Thus, in one aspect the invention provides a process forisolating a large quantity (e.g. gram or kg amounts) of plasmid DNA. Themethod involves the steps of: (a) lysing cells containing the plasmidDNA with a lysis agent, thereby forming a lysate; (b) treating thelysate with a high salt agent that preferably is capable of forming aprecipitable complex with non-plasmid DNA cellular components containedin the lysate, thereby forming a treated solution; and (c) purifying thetreated solution to provide isolated plasmid DNA.

[0025] The phrase “plasmid DNA” is meant to include all forms of plasmidDNA, such as supercoiled plasmid DNA (type I), nicked circle plasmid DNA(type (II), and linearized plasmid DNA (type III), as well as denaturedplasmid DNA. The plasmids may include any of a wide variety of vectors,origins of replication, and genetic elements (such as selectable genes,polylinkers, promoters, enhancers, therapeutic genes, leader peptidesequences, introns, polyadenylations signals, etc.) known to thoseskilled in the art. Genes encoding diverse proteins (includingtherapeutic proteins such as IL-2, IL-12 and the like) may be insertedinto the plasmid, and the genes may constitute, for example, genomicDNA, cDNA, synthetic DNA, and polynucleotide and oligonucleotidesequences.

[0026] By “lysis agent” is meant any agent capable of breaking open acell containing plasmid DNA and thereby releasing the contents of thecell. The lysis agent preferably is alkali or basic, i.e., it has a pHabove 7.0, preferably about 12 to 13. Many suitable agents are known tothose in the art, for example, a solution containing 0.2N sodiumhydroxide in 1% SDS. Other concentrations of sodium hydroxide in SDS mayalso be used. Other detergents (tween, np40, sarkosyl, etc.) such asnon-ionic detergents (e.g., triton X) and lysozyme plus heat treatmentmay all also be used.

[0027] By “high salt agent” is meant a substance that is capable ofprecipitating out a significant portion (preferably at least 25%, morepreferably at least 50%, most preferably at least 75% or 90%) of any RNAmolecules released from the cells. The high salt agent preferably hasone or more salts at a pH above approximately 5.5, for example a mixtureof 1M potassium acetate and 7M ammonium acetate at a pH between 7.0 and9.0. It may be possible to replace the potassium acetate with apotassium agent or ion and/or KCl, NaCl, or NH₄OAc. The lysate ispreferably treated with the high salt agent for at least six hours atapproximately four degrees Celsius.

[0028] In preferred embodiments, the process does not involve the use ofRNase, the process yields isolated plasmid DNA that ispharmaceutical-grade plasmid DNA suitable for administration to humans,and at least 100 milligrams of the isolated plasmid DNA is obtained,more preferably at least 1 g, most preferably at least 10 g, 10 g or1,000 g (1 kg). In other preferred embodiments, the invention provides aprocess for isolating plasmid DNA involving the steps of: (a)resuspending cells in approximately 50 mM of Tris-HCl buffer at a pH ofabout 8.0 and approximately 10 mM EDTA(Na)₂; (b) lysing cells containingthe plasmid DNA with a lysis agent comprising an approximately equalvolume of 0.2N sodium hydroxide in 1% SDS, thereby forming a lysate; (c)treating the lysate with a high salt agent that comprises a mixture of1M potassium acetate and 7M ammonium acetate at a pH between 7.0 and9.0, preferably capable of forming a precipitable complex withnon-plasmid DNA cellular components contained in the lysate, therebyforming a treated solution; and (d) purifying the treated solution toprovide isolated plasmid DNA. Step (d) may involve subsequentlyisolating from the supernatant of the cellular lysate a sample of highlypurified supercoiled plasmid DNA.

[0029] In view of the above, it can be seen that, among other things,the invention provides an improved process for isolating plasmid DNAfrom alkaline lysates of a cell containing the plasmid DNA by treatingthe lysate with a high salt agent, preferably capable of forming aprecipitable complex with non-plasmid DNA cellular components containedin the lysate.

[0030] In another aspect, the invention provides a process for isolatingplasmid DNA involving the steps of: (a) lysing cells containing theplasmid DNA with a lysis agent, thereby forming a lysate; and (b)purifying the lysate with anion exchange chromatography using a stepgradient, thereby producing isolated plasmid DNA enriched with at least80% supercoiled plasmid DNA.

[0031] In preferred embodiments the anion exchange chromatography isperformed with a resin having a particle size of 20-40 microns, theanion exchange chromatography has a plasmid DNA binding capacity ofabout 1.5 mg of plasmid per mL of resin, more preferably a plasmid DNAbinding capacity of about 3 (or more) mg of plasmid per mL of resin, andthe anion exchange chromatography is performed with a Fractogel EMD TMAE(650-S) resin. Thus, the invention provides an improved process forisolating plasmid DNA from lysate of cells containing the plasmid DNA bypurifying the lysate with anion exchange chromatography using a stepgradient, thereby producing isolated plasmid DNA enriched with at least80% supercoiled plasmid DNA.

[0032] In another aspect, the invention features a process for isolatingplasmid DNA involving the steps of: (a) lysing cells containing theplasmid DNA with a lysis agent, thereby forming a lysate; and (b) usinghydrophobic interaction chromatography (HIC) to purify the lysate,thereby providing isolated plasmid DNA. Typically the lysate is notapplied directly to the HIC column directly, but is first sent throughan anion exchange chromatography resin, such as the Fractogel TMAE resindiscussed herein.

[0033] In preferred embodiments the hydrophobic interactionchromatography is performed with at least 1.6M ammonium sulfate (at 2.OMammonium sulfate the supercoiled plasmid binds to the HIC column), Tris,and EDTA, and the hydrophobic interaction chromatography is performedwith an Octyl Sepharose 4 FF resin. This is a flow through column thatallows supercoiled plasmid to flow through the column while RNA, andchromosomal DNA endotoxin and denatured DNA binds to the resin. Thecolumn is moderately hydrophobic due to octyl alkyl chains as its ligandon agarose beads. Other resins having similar properties such as phenylsepharose, butyl sepharose, and others, may also be used.

[0034] In other preferred embodiments the plasmid DNA is notprecipitated and the process involves no linear gradients and uses noorganic solvents, the isolated plasmid DNA is substantially free ofendotoxins (for example, 10 to 100 Endotoxin Units per mg of plasmid DNAas measured with a Limulus Amebocyte Lysate Assay) and host cellchromosomal DNA (for example, about 1% to 3% as measured by LCR), theplasmid DNA is not exposed to acidic pH or elevated temperatures (e.g.,about pH 8 and 22° C.), the isolated plasmid DNA is produced in a yieldof at least 60% (preferably 70%, more preferably 80%, most preferably90%), the hydrophobic interaction chromatography is performed in anaqueous solution containing a high concentration of salt, such as 1.6Mammonium sulfate, and the cells are recombinant E. coli cells. Thus, theinvention features an improved process for isolating plasmid DNA from alysate of a cell containing the plasmid DNA by using hydrophobicinteraction chromatography to purify the lysate, thereby providingisolated plasmid DNA. Preferably the lysate is first partially purifiedwith anion exchange chromatography, for example with a TMAE resin, andthen the anion exchange chromatography pool, (for example, a TMAE pool)is further purified with HIC.

[0035] In yet another aspect, the invention provides a device forisolating plasmid DNA from cells containing the plasmid DNA, comprising:(a) a means for providing cell resuspension in a semi-continuous mode;(b) a means for providing mixing and cell lysis in a continuous flowmode; and (c) a means for providing chilling and mixing to denature andprecipitate chromosomal DNA, protein, and RNA.

[0036] In preferred embodiments, the means for providing efficient andgentle mixing and cell lysis in a continuous flow mode comprises animpeller mixer, an in-line static mixer, and/or a lysis coil. The devicepreferably has a means for performing hydrophobic interactionchromatography and the means for providing fast chilling and efficientand gentle mixing to denature and precipitate chromosomal DNA, protein,and RNA preferably comprises a chilled jacketed tank.

[0037] Other and further objects, features, and advantages will beapparent from the following description of the drawings and thepresently preferred embodiments of the invention, as well as theexamples provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 is a schematic process diagram including resuspension andlysis (in line mixer), and neutralization (impeller mixer).

[0039]FIG. 2 is a flow chart for Process I.

[0040]FIG. 3 is a flow chart for Process II.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The invention identified here is a novel, scalable, andalternative alkaline lysis process for large scale plasmid DNAisolation. The present invention involves a series of new unitoperations and devices for cell resuspension, cell lysis, andneutralization. The invention can provide controllable and efficientoperation and consistent performance and is economical. Described beloware various preferred methods for performing the invention as well as adetailed description of the devices and certain aspects of theinvention.

[0042] The most preferred embodiments are described immediately below,followed by a discussion of several of the steps and devices that may beuseful in practicing the invention.

[0043] One of the preferred embodiments involves the use of a continuousflow device with in-line mixing for resuspending bacterial cells, lysingwith alkali and detergent, and neutralizing the lysate. This has severaladvantages (relative to batch operation in multiple bottles or largetanks), including: (1) better cell resuspension and mixing of solutions;(2) better control of lysis times; and (3) lower shear, proving lesschromosomal DNA contamination (very significant).

[0044] A second preferred embodiment involves the use of a modified,non-acidic neutralization solution that can be added to analkaline/detergent lysate to precipitate RNA as well as chromosomal DNA,protein, lipids, etc. from the lysate. Some of the advantages (relativeto 3M potassium acetate, pH-5) are: (1) removes majority of RNA(especially high molecular weight) prior to column chromatography (verysignificant); (2) eliminates exposure of plasmid DNA to damaging acid;(3) eliminates need to partially purify plasmid prior to RNAprecipitation.

[0045] A third preferred embodiment involves the use of Fractogel EMDTMAE tentacle ion exchange resin to separate supercoiled plasmid fromopen circular plasmid and from protein and low molecular weight RNA.Here, the advantages (relative to Sepharose Q or other low pressure ionexchange resins) include: (1) higher capacity (1.5 mg plasmid/mL resin);(2) high flow rate; (3) able to separate supercoiled and open or nickedcircular plasmid using a simple, convenient, scalable salt step gradient(very significant).

[0046] A fourth preferred embodiment involves the use of hydrophobicinteraction chromatography (HIC) to separate plasmid DNA fromchromosomal DNA, RNA, protein, endotoxin, and denatured plasmid DNA. Theadvantages of this embodiment include: (1) removes E. coli DNA (verysignificant); (2) removes residual RNA, endotoxin, and denatured plasmidDNA; (3) optionally separates supercoiled from nicked or open circularplasmid.

[0047] The fifth preferred embodiment is an integrated processesemploying some or all of the above (Process I, Process II, and potentialvariants).

[0048] Alkaline Lysis

[0049] Conventional alkaline lysis techniques involve the use of 3Mpotassium acetate at a pH of about 5.5. A significant improvementpresented herein is the use of high concentrations of unbuffered salt.(A number of monovalent salts are suitable and some divalent salts willalso have acceptable solubility properties.) For example, by adding avolume of an unbuffered solution of 1M potassium acetate and 7M ammoniumacetate and letting the mixture stand for at least 2 (preferably atleast 6) hours at 4 degrees Celsius a surprising and unexpected resultis achieved. Namely, not only is denatured chromosomal DNA, protein, andcellular debris trapped in a precipitable salt/detergent complex, but asignificant portion of RNA is also precipitated, allowing for theelimination of an RNase treatment step. The potassium acetate preferablyis present in an excess in order to aid in the precipitation the SDS DNAcomplex. Furthermore, the RNA that remains in solution is typically lowmolecular weight and can be separated from plasmid DNA by subsequentchromotographic steps (e.g., anion exchange, HIC, size exclusion, etc.).At lower dilutions to the TMAE load (1.5 vs. 3 or 4 volumes), themajority of RNA that remains flows through the TMAE column.

[0050] Anion Exchange Chromatography

[0051] The present invention makes use of anion exchange chromatography,preferably with a resin such as Fractogel EMD TMAE(s) 650-S having a20-40 micron particle size. This advantageously provides a high DNAbinding capacity of about 3 mg/mL, preferably about 1.5 mg/mL and allowsfor the efficient removal of proteins, RNA, low molecular weightmolecules, and probably some chromosomal DNA. Surprisingly, thisinvention allows for the enrichment of supercoiled form of plasmid DNAby over 80% using a step gradient. Enrichment of supercoiled plasmid isachieved by removal of more open circular plasmid than is achieved inthe preferred embodiment of Process I outlined in FIG. 2. The percentageof enrichment may be measured with a 1% reliant gel (FMC), 0.8% agarosegel electrophoresis, and may also be measured using an ion exchange HPLCassay using, for example, a DEAE column.

[0052] Hydrophobic Interaction Chromatography

[0053] Hydrophobic interaction chromatography (“HIC”) is generallypracticed by binding compounds of interest in an aqueous solutioncontaining high concentrations of salt (e.g. ammonium sulfate). Elutionis accomplished by lowering the salt concentration. No volatile orflammable organic solvents are used, and no organic modifiers like TEAAare generally required.

[0054] There are a variety of HIC resins which are commerciallyavailable, differing in both backbone and functional chemistries. Ingeneral, they can be made to work for plasmid DNA purification, howeverthere are some which are preferred. The HIC step is a flow through step,i.e., the supercoiled and open circular plasmid flow through the columnwhile RNA, chromosomal DNA, denatured plasmid DNA and endotoxins areretained on the column.

[0055] The HIC resins are initially equilibrated in high ionic strengthmultivalent salts such as ammonium sulfate. These conditions enhancehydrophobic interaction and thus retention of various biomolecules. Byreducing the salt concentration the hydrophobic interaction is lessenedand the biomolecules begin to elute from the resin depending on theirhydrophobicity. In the case of plasmid DNA, it has been observed thatthe relaxed open circle form has very little interaction with HIC resinsin the presence of high salt. While under these conditions thesupercoiled form does interact and is retained by the resin leading to aresolution of the forms. As the salt concentration is lowered, thesupercoiled form is eluted, while the RNA, chromosomal DNA and denaturedplasmid DNA remain bound. Thus, the supercoiled plasmid may be purifiedfrom undesired plasmid forms (open circular, denatured) and fromcontaminants (chromosomal DNA, RNA, protein, endotoxin) using HIC. Thisis an unexpected result.

[0056] The interaction of biomolecules with the HIC resins can also bemodified as a function of the multivalent salt selected. It has beenobserved that in general sodium citrate and potassium phosphate saltscan increase hydrophobic interaction of plasmid DNA greater thanammonium sulfate. Thus, by optimizing the selection of resin and saltconditions, one can affect the quality of purification for a givenplasmid construct.

[0057] Devices

[0058] All devices described in this invention were built to large scale(process capacity is greater than 2 kg of wet cell paste) to demonstratethe feasibility and effectiveness of the invented devices and operationprocedures, and the concept of continuous-flow-through resuspension,lysis, and neutralization. A polypropylene or stainless steel conicaltank with a bottom three-way valve was connected to two sets ofplatinum-cured silicone tubing. Those skilled in the art will recognizethat many other types of containers and/or connections may besubstituted without substantially changing the function, mode ofoperation or result obtained the overall device. One set of tubingcontained one unit of ½″ in-line static mixer and two units of ⅜″in-line static mixers. These in-line mixers are for resuspending cellsand homogenizing cell concentration. Other sizes and numbers of mixersmay also suitably be used. The other set of tubing is used to transferresuspended cells to contact lysis solution.

[0059] A MasterFlex peristaltic transfer pump was used for resuspensionand homogenization. Other suitable (exp-low shear) pumps are availableand may be substituted without materially altering the overall device. A43 ft×1″ OD stainless steel tubing was coiled with approximately 2′diameter and a 6″ pitch and used as the lysis coils. Those skilled inthe art will recognize that the exact length, diameter, and pitch may bevaried by at least 10% and perhaps even over 50% without departing fromfunctional lysis goal served by the coils. The bottom of the lysis coilswas connected to a 1″ in-line static mixer (multiple mixers may be used)which is used to mix resuspended cells and lysis solution. A MasterFlexperistaltic pump with two stacked pump heads is used to transferresuspended cells and lysis solution to the 1′ in-line static mixer formixing. As noted above, other pumps and mixers may also be used.

[0060] The holding tank for precipitation is a 150 L stainless steeljacketed tank. Other suitable containers will preferably have a volumeof at least 100-200 L or more. The tank jacket was connected to arecirculating chiller which has the ability to chill the coolant to −15°C. Other chilling means may be substituted and preferably have theability to achieve at least 10 degrees Celsius, more preferably zerodegrees or lower. Since two approaches were adopted to mix lysate andneutralization solution, two sets of operations and devices were used toaccomplish the mixing of lysate and neutralization solution. In thefirst approach, the effluent of the lysis coils went directly into thechilled tank. An impeller mixer was used to mix lysed cells andneutralization solution inside the tank. The second approach involves a1″ or 2″ (or other conveniently sized) in-line static mixer. Lysed cellsand neutralization solution were pumped into the in-line static mixersimultaneously (one skilled in the art will recognize that cells orlysis solution could be pumped first, followed by the other), and theeffluent of the mixer was directed into chilled tank for precipitation.

[0061] It is possible to run the EM Merk Fractogel EMD 650(s) TMAEcolumn in a displacement mode where super coiled plasmid displaces opencircular plasmid off the resin, thereby enriching for super coiledplasmid. The column is equilibrated in 0.5M NaCl, TE buffer, the load(neutralized lysis supernatant process 2) is diluted with 1.5 volumesWPI then loaded onto the column. The column is loaded at 3.0 mg/mlresin. The last quarter of the flow through of the load to the TMAEcolumn is recirculated over the column to allow SC plasmid to competeoff open circular plasmid. The column is then washed to baseline with0.5M NaCl, TE after the recirculation has finished. The plasmid is theneluted with 1.9M (NH₄)₂SO₄.

[0062] Another resin from EM that displays similar properties. The resinis a Fractogel (R) EMD TMAE Hicap (M). This is a high capacity resinwith better flow characteristics than the (S) TMAE resin. This Hicapresin may allow binding plasmid between 3 and 5 mg/ml of resin and allowfor the enrichment of SC plasmid at a very high level.

EXAMPLES

[0063] The following examples are solely for illustrative purposes andare not meant to limit the scope of the invention.

Example 1 Process I

[0064] The step-by-step methodology of Process I is outlined in FIG. 3.

[0065] Step 1 involves fermentation, cell harvesting, and washing. Theseare well known procedures that may be carried out in wide variety ofways known to those skilled in the art.

[0066] Step 2 involves alkaline lysis and neutralization. This stepextracts plasmid, chromosomal DNA, RNA and protein. As explained indetail above, use of a high concentration of buffered salts ispreferable.

[0067] Step 3 involves centrifugation and filtration. These also arewell known techniques that may be performed in a variety of ways knownto those skilled in the art.

[0068] Step 4 is RNase treatment at 37 degrees Celsius for one hour.This step may optionally be removed (as in Process II described inExample 2 below) by utilizing the preferred alkaline lysis and high saltagents described herein.

[0069] Step 5 involves filtration and dilution with 2 volumes of WFI.These are standard techniques that may easily be modified by thoseskilled in the art.

[0070] Step 6 involves the use of a Q Sepharose HP column to removeprotein, and some RNA and chromosomal DNA. Other Q resins may also work,or a Fractogel TMAE resin may be used as in Process II below.

[0071] Step 7, involves the use of DEAE 650-S to remove RNA.

[0072] Step 8 uses Phenyl 650-S to remove chromosomal DNA, endotoxin,denatured plasmid, and some RNA. Other phenyl resins and 8 carbon octylresins may also be used.

[0073] Steps 9 and 10 are ultrafiltration/diafiltration and sterilefiltration to yield the final product. These are common procedures thatmay be insubstantially modified by those skilled in the art withoutdeparting from the scope of the invention.

Example 2 Process II

[0074] The steps of Process II are outlined in FIG. 4. Step (a) isfermentation of plasmid containing E. coli, cell harvesting, e.g. bycentrifugation and washing and resuspending cells as in step 1 ofProcess I.

[0075] Step (b) is alkaline lysis (e.g., 1% SDS and 0.2N NaOH) and highsalt precipitation to extract plasmid and remove chromosomal DNA andsome RNA and protein. The high salt precipitation may involve adding 1Mpotassium acetate and 7M ammonium acetate (concentrations of both may bevaried, with higher concentrations of ammonium acetate being better forRNA precipitation) and holding the temperature at 4 degrees Celsius forapproximately 6 to 12 hours to precipitate RNA (times and temperaturesmay be varied; lower temperatures and longer times improve precipitationand incubation at 4 degrees Celsius provides a higher percentage ofprecipitation). The ability to precipitate RNA during neutralization ofthe alkaline lysate helps eliminate the need for RNase. The acetatesolution does not need to be pH adjusted and the pH is approximately 8.Conventional techniques using 3M potassium acetate at pH 5 aredramatically less effective at RNA precipitation and provide greaterrisk of DNA damage.

[0076] Step (c) is centrifugation/filtration and 1.5 volume dilutionwith water for irrigation (WFI).

[0077] Step (d) involves the use of, for example, Fractogel EMD TMAE650(s), a strong anionic change resin, to remove residual protein andsome RNA and chromosomal DNA and enrich the supercoiled form of theplasmid DNA. This resin is capable of separating nicked/relaxed plasmidfrom supercoiled plasmid with a preparative, low pressure, step elution.Variations in particle size and chemistry may be acceptable. The nextprocedure is to wash the nicked and/or relaxed circular plasmid, as wellas residual RNA, off of the resin with ˜0.6M NaCl (10 mM Tris, 1 mMEDTA, pH 8.0). The supercoiled plasmid thus remains bound to resin.Other salts may also be suitable at an appropriate concentration(depending on the salt). This is followed by eluting the covalentlyclosed supercoiled plasmid off of the resin with about 1.9M ammoniumsulfate (A.S.). Elution with concentrated A.S. allows eluate to flowthrough the next column. Elution with ≧0.7M NaCl is also suitable, butin that case the eluate must be adjusted to ˜1.6 M.A.S. prior to thenext column. Other salts may also be suitable at an appropriateconcentration (depending on the salt).

[0078] Step (e) involves the use of Octyl Sepharose 4FF for removal ofchromosomal DNA, RNA, endotoxin, and denatured plasmid. This involvespassing the TMAE pool through an Octyl Sepharose 4 Fast Flow (Pharmacia#17-0946) resin and collecting the flow through fraction. Otherhydrophobic interaction (HIC) resins may also be used (e.g. PharmaciaPhenyl Sepharose, Pharmacia Butyl Sepharose, similar resins from othervendors). At ˜1.6M. ammonium sulfate, plasmid flows through and residualRNA, endotoxins, and E. coli DNA bind. Alternatively, one can bind theTMAE pool in the presence of ˜2.0M. ammonium sulfate, which allowsrelaxed and/or nicked circular plasmid to flow through, while thesupercoiled plasmid binds; the supercoiled can then be eluted with ≦1.6Mammonium sulfate. Variations in resin particle size, etc., may beacceptable and some alternative salts are also suitable at appropriateconcentrations.

[0079] Steps (f) and (g). are ultrafiltration/ diafiltration followed bysterile filtration to yield final product.

[0080] Some of the advantages of Process II are listed below: (1) Canenrich/resolve for supercoiled plasmid DNA; (2) Plasmid DNA is neverprecipitated; (3) No linear gradients necessary; (4) No organic solventsused; (5) In preferred embodiments in Process I, 1.62 volumes of lysissolution (neutralizing) is used whereas in preferred embodiments ofProcess II, 1 volume of lysis solution (high salt 1M potassium acetateand 7M NH₄OAc) is used; (6) No RNase required; (7) Significantly reducesendotoxin levels; (8) DNA is not exposed to either acid pH or elevatedtemperatures; (9) Scaleable for Industrial Quantities; and (10) Highplasmid DNA yields (60-90%) and high purity (90-95%).

Example 3 Unit Operations of the Invention

[0081] The unit operations in this invention are in-line static mixers,flow through lysis coils, and a chilled jacketed tank.

[0082] The cell paste, either from direct isolation from fermentation orthawed if frozen, was resuspended in TE buffer (50 mM Tris and 10 mMEDTA) by circulating the cell-buffer mixture through the in-line staticmixer in a pump-around mode. The flow rate was specifically determinedto be capable of effectively resuspending cells without breaking them.

[0083] The resuspended cells were mixed with the lysis solution (200 mMNaOH and 1% SDS) by flowing through the in-line static mixer, thenentering the lysis coils for continuous lysis at a constant rate. Theoptimal flow rate to deliver cell-lysis solution mixture through thestatic mixer and lysis coils was determined to maximize the flow rate,minimize the shear force, and generate homogeneous mixture and desiredmolecule residence time.

[0084] The lysate was mixed with cold neutralization solution (1.85MKOAc, 1.16N NH₄OAc, and 1.15M NaCl pH 5.5 Process I buffer solution) todenature and precipitate high molecular weight host chromosomal DNA,host proteins, and host RNA. The mixing of neutralization solution andlysate is either accomplished by flowing through the in-line staticmixer in a continuous mode, or by agitating the low speed impeller mixerin a batch mode.

[0085] After mixing, the acidified lysate will be retained in thechilled jacketed tank for 20-30 minutes to complete the precipitationand denaturation.

Example 4 Scale-Up Test

[0086] The scale-up test was started from transferring thawed cells andTE buffer into the conical tank. The cell-TE mixture was then pumpedthrough the in-line static mixer to the optimized flow rate. Aftercompleting cell resuspension, cells and lysis solution were pumpedthrough another in-line static mixer simultaneously for mixing. At thesame time of mixing cells and lysis solution, cells remained in theconical tank were still circulated through the resuspension-in-linestatic mixer to ensure the homogeneity of cell pool to maximize theeffectiveness of alkaline lysis. Lysate continuously flowed through thelysis coils at a fixed residence time.

[0087] The effluent from the lysis coils was directed to a chilledjacketed tank containing a high salt neutralizing solution and equippedwith an impeller stirrer for mixing for mixing. However, the preferredmethod would be to mix the highly viscous alkaline cell lysis solutionwith the much lower viscosity high salt solution using an in-line staticmixer to minimize the shearing of the chromosomal DNA. This would allowfor instantaneous mixing of the two solutions in a gentle manner to formthe denatured chromosomal DNA/denatured protein/sodium dodecyl sulphatecomplex. Afterwards the neutralized precipitated lysate would bedirected to a cold jacketed holding tank.

Example 5 Isolation of Plasmid

[0088] 1.5 kg (wet weight) of frozen recombinant E. coli cells harboringa plasmid was thawed and TE buffer was added to make a total volume to10.5 L. The mixture of cell paste and TE buffer was circulating throughthe in-line mixers at 3L/min for 5-10 minutes at room temperature toobtain homogeneous cell concentration. The resuspended cells and roomtemperature lysis solution were pumped to the 1″ in-line static mixersimultaneously at an overall flow rate of 1.0 L/min. The mixture ofcells and lysis solution continuously flowed through the lysis coils at1.0 L/min.

[0089] The lysate exiting from the lysis coils directly into thejacketed tank which contained 17 L cold neutralization buffer. Thelysate and cold neutralization solution were gently mixed by an impellermixer to avoid shearing the host chromosomal DNA. The acidified lysatewas kept in the chilled-jacketed tank for about 30 minutes to completelyprecipitate the host chromosomal DNA, host proteins, and host RNA.

[0090] After completing the precipitation, plasmid was isolated andpurified. The host chromosomal DNA level in the final product wasassayed at 0.4%.

Example 6 Reproducibility of the Alkaline Lysis Process

[0091] 1.5 kg (wet weight) of frozen recombinant E. coli cells harboringa plasmid was used to examine the reproducibility of the inventedprocess. All buffers, devices, and procedures were adopted to mimicExample 5. The isolation and purification procedures were also the sameas that used in Example 5. The host chromosomal DNA level in the finalproduct was assayed at 0.12%. Example 7: Variability of Final Productquality Without Use of With Use of Device Device Number of Lots Used for37 7 Comparison Average Chromosomal DNA, % 2.83 0.63 Standard Deviation2.68 0.64

[0092] The above showed that the use of the device is superior in regardto both final product quality (chromosomal DNA level) and processperformance consistency.

Example 8 Purification of Plasmid DNA Containing the α₁-Antityrpsin Gene

[0093] Previous purification of an α₁-antitrypsin plasmid prep had beendone by the process 1 protocol (FIG. 1) in which the H.I.C. step had notyet been incorporated. Thus the purification process ended after theDEAE column step and the resulting plasmid pool was concentrated bystandard ethanol precipitation. The final product of α₁-antitrypsinplasmid prepared by the above method still contained unacceptable highlevels of chromosomal DNA (bacterial), RNA, denatured plasmid, andendotoxins.

[0094] Therefore a phenyl hydrophobic interaction chromatography (HICfrom TosoHaas) step was added to the process 1 protocol following theDEAE chromatography column in order to reduce the contaminants to a muchlower level and thereby increasing the purity of the plasmid DNA to alevel ≧95%.

[0095] Approximately 315 grams of D-5α E. coli cells (wet weight)containing the α₁-antitrypsin plasmid was exposed to alkaline lysis for5 minutes and the denatured chromosomal DNA/protein/sodiumdodecylsulfate complexes were precipitated by addition of a saltsolution containing 1.85M potassium acetate, 1.15M sodium chloride,1.16M ammonium acetate at pH 5.5. After centrifugation, filtration, andRNAse treatment the resulting supernatant (8L) contained ˜35 μg plasmidDNA/mL.

[0096] Following anion exchange chromatography of the lysate on both QSepharose 4FF (Pharmacia) and DEAE 650-S (TosoHaas) which removesprotein, RNA and the bulk of chromosomal DNA, the plasmid pool was thenpumped onto a phenyl 650-S (TosoHaas) HIC resin.

[0097] The phenyl 650-S resin (100 mL) was packed into a 5 cm diametercolumn to a height of 5cm. The column was equilibrated withapproximately 500 mL of 1.75M (NH₄)₂SO₄ at 20 mL/minute. Then 350 mL ofthe DEAE elution pooly containing the α₁-antitrypsin plasmid was dilutedwith an equal volume of 3.5M (NH₄)₂SO₄ which results in a finalconcentration of 1.75M (NH₄)₂SO₄. The entire 700 mL of plasmid DNA in1.75M (NH₄)₂SO₄ was pumped through the HIC column and the flow throughpeak which contains the α₁-antitrypsin plasmid was saved for furtherprocessing by ultrafiltration and diafiltration (UF/DF). The bound RNA,chromosomal DNA< denatured plasmid and endotoxins were eluted in aMilli-Q water wash followed by a 70% ethanol (v/v) wash.

[0098] A Pellicon-2 system (Millipore) containing a 50 kD casseter(Millipore) was used to UF/DF the HIC flow through containing theα₁-antitrypsin plasmid DNA in 1.75M (NH₄)₂SO₄. The final product(α₁-antitrypsin plasmid) was concentrated 7.5-fold by UF, anddiafiltered against 10 diavolumes of Milli-Q water.

[0099] Analysis of the final product, α₁-antitrypsin plasmid, indicatedthe preparation 95% plasmid DNA, <5%RNA, 0.05% E. coli DNA, <0.06%protein, <0.2Eu//mg of plasmid DNA for endotoxins, and complete removalof denatured plasmid DNA. This represents a reduction by the HIC resinin bacterial chromosomal DNA of between 10 and 100-fold from whattypically contaminates plasmid preps after the DEAE anion chromatographystep. In addition, RNA was reduced between 1 and 2-fold, endotoxinlevels were lowered at least 250-fold, both protein and denaturedplasmid levels have been reduced to essentially zero.

[0100] It should also be pointed out that most HIC resins will bindsupercoiled DNA at ≧2M (NH₄)₂SO₄ while the open circle form will notbind under these conditions. This allows for a significant enrichment ofthe supercoiled plasmid DNA in the final product. Thus HIC resins inconjunction with anionic-exchange resins offer the ability atlarge-scale to remove a substantial amount of contaminants whichcopurify with the plasmid DNA on ion-exchange resins. In addition, HICresins can be used to selectively purify supercoiled plasmid DNA.

Example 9 A Hydrolysis Assay

[0101] This assay is used to quantify the amount of RNA present in asolution of plasmid DNA.

[0102] Standard Curve

[0103] (1) Use E. coli rRNA as standards from Sigma; ribonucleic acid,ribosomal from E. coli, Strain W; Cat. No. R-7628). Make up rRNA toconcentrations of 0, 10, 20, 30, 40 and 50 μgs/ml by diluting theresuspended RNA in water.

[0104] (2) Run 367 l of each standard through RNA hydrolysis procedure(see below).

[0105] (3) Plot standards with concentration versus A260 and curve fitwith a linear fit, forcing the curve through zero.

[0106] (4) Use the slope from the curve to calculate sample RNAconcentrations.

[0107] Hydrolysis Procedure

[0108] (1) Take 467 μls of sample. If necessary, dilute before taking467 μls so that RNA is less than 50 μgs/ml. Put samples in screw captubes so that tubes do not pop open during heating.

[0109] (2) Add 33 μl 1.5M KOH to bring volume to 0.5 mls.

[0110] (3) Heat at 110° C. for 1 hour. The heat and alkali willhydrolyze the RNA.

[0111] (4) Turn off heat block after one hour, leaving the samples inthe block, and allow samples to cool to less than 100° C. (about 10minutes).

[0112] (5) Remove from heat block and add 32 μls of 10% HCl.

[0113] (6) Add 968 μls 100% ethanol to bring volume to 1.5 mls. Theabsolute ethanol will precipitate the DNA in the sample.

[0114] (7) Place the samples at ˜20° C. for at least 20 minutes.

[0115] (8) Centrifuge samples at 4° C. for at least 15 minutes at 14,000rpm.

[0116] (9) Blank UV spectrophotometer with WFI, water for injection orMilliQ water. Measure A260 and A232 of supernatants.

[0117] (10) Use A260 to calculate RNA concentration with the standardcurve and, if necessary, use A232 to adjust for protein.

[0118] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themethods, procedures, treatments, molecules, specific compounds describedherein are presently representative of preferred embodiments areexemplary and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention aredefined by the scope of the claims. It will be readily apparent to oneskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

[0119] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A process for isolating plasmid DNA comprisingthe steps of: (a) lysing cells containing said plasmid DNA with a lysisagent, thereby forming a lysate; (b) treating said lysate with a highsalt agent, thereby forming a treated solution; and (c) purifying saidtreated solution to provide isolated plasmid DNA.
 2. The process ofclaim 1 , wherein said plasmid DNA is a mixture of supercoiled plasmidDNA, nicked circle plasmid DNA, and linearized plasmid DNA.
 3. Theprocess of claim 1 , wherein said process does not involve the use ofRNase.
 4. The process of claim 1 , wherein said high salt agent iscapable of precipitating a significant portion of any RNA molecules fromsaid cells.
 5. The process of claim 1 , wherein said high salt agentcomprises one or more salts at a pH above approximately 5.5.
 6. Theprocess of claim 5 , wherein said high salt agent comprises a mixture of1M potassium acetate and 7M ammonium acetate at a pH between 7.0 and9.0.
 7. The process of claim 6 , wherein said lysate is treated withsaid high salt agent for at least six hours at approximately fourdegrees Celsius.
 8. The process of claim 1 , wherein said isolatedplasmid DNA is pharmaceutical-grade plasmid DNA suitable foradministration to humans.
 9. The process of claim 1 , wherein at least100 milligrams of said isolated plasmid DNA is obtained.
 10. A processfor isolating plasmid DNA comprising the steps of: (a) resuspendingcells in approximately 50 mM of Tris at a pH of about 8.0 andapproximately 10 mM EDTA(Na)2; (b) lysing cells containing said plasmidDNA with a lysis agent comprising an approximately equal volume of 0.2Nsodium hydroxide in 1% SDS, thereby forming a lysate; (c) treating saidlysate with a high salt agent comprises a mixture of 1M potassiumacetate and 7M ammonium acetate at a pH between 7.0 and 9.0, therebyforming a high salt solution; and (c) purifying said treated solution toprovide isolated plasmid DNA.
 11. The process of claim 10 , wherein saidisolated plasmid DNA is pharmaceutical-grade plasmid DNA suitable foradministration to humans.
 12. The process of claim 10 , wherein at least100 milligrams of said isolated plasmid DNA is obtained.
 13. In theprocess of claim 10 for isolating plasmid DNA from lysate of a cellcontaining said plasmid DNA, wherein the improvement comprises treatingsaid lysate with a high salt agent capable of precipitating non-plasmidDNA cellular components contained in said lysate.
 14. The process ofclaim 13 , wherein said high salt agent is capable of precipitating asignificant portion of any RNA molecules from said cells.
 15. Theprocess of claim 13 , wherein said high salt agent comprises one or moresalts at a pH above approximately 5.5.
 16. The process of claim 15 ,wherein said high salt agent comprises a mixture of 1M potassium acetateand 7M ammonium acetate at a pH between 7.0 and 9.0.
 17. The process ofclaim 16 , wherein said lysate is treated with said high salt agent forat least six hours at approximately four degrees Celsius.
 18. A processfor isolating plasmid DNA comprising the steps of: (a) lysing cellscontaining said plasmid DNA with a lysis agent, thereby forming alysate; and (b) purifying said lysate with anion exchange chromatographyusing a step gradient, thereby producing isolated plasmid DNA (Theprocess of claim 18 , wherein the isolated plasmid DNA is enriched withat least 80% supercoiled plasmid DNA).
 19. The process of claim 18 ,wherein said isolated plasmid DNA is pharmaceutical-grade plasmid DNAsuitable for administration to humans.
 20. The process of claim 18 ,wherein at least 100 milligrams of said isolated plasmid DNA isobtained.
 21. The process of claim 18 , wherein said anion exchangechromatography is performed with a resin having a particle size of 20-40microns.
 22. The process of claim 22 , wherein said anion exchangechromatography has a plasmid DNA binding capacity of about 1.5 mg ofplasmid per mL of resin.
 23. The process of claim 18 , wherein saidanion exchange chromatography is performed with a Fractogel EMDTMAE(650-S) resin.
 24. The process of claim 18 for isolating plasmid DNAfrom lysate of a cell containing said plasmid DNA, wherein theimprovement comprises purifying said lysate with anion exchangechromatography using a step gradient, thereby producing isolated plasmidDNA enriched with at least 80% supercoiled plasmid DNA.
 25. The processof claim 24 , wherein said anion exchange chromatography is performedwith a resin having a particle size of 20-40 microns.
 26. The process ofclaim 24 , wherein said anion exchange chromatography has a plasmid DNAbinding capacity of about 1.5 mg of plasmid per mL of resin.
 27. Theprocess of claim 24 , wherein said anion exchange chromatography isperformed with a Fractogel EMD TMAE(S) resin.
 28. A process forisolating plasmid DNA comprising the steps of: (a) lysing cellscontaining said plasmid DNA with a lysis agent, thereby forming alysate; and (b) using hydrophobic interaction chromatography to purifysaid lysate, thereby providing isolated plasmid DNA.
 29. The process ofclaim 27 , wherein said hydrophobic interaction chromatography isperformed with at least 1.6M ammonium sulfate.
 30. The process of claim28 , wherein said hydrophobic interaction chromatography is performedwith an Octyl Sepharose 4 FF resin.
 31. The process of claim 28 ,wherein said isolated plasmid DNA is pharmaceutical-grade plasmid DNAsuitable for administration to humans.
 32. The process of claim 28 ,wherein at least 100 milligrams of said isolated plasmid DNA isobtained.
 33. The process of claim 28 , wherein said plasmid DNA is notprecipitated and wherein said process involves no linear gradients anduses no organic solvents.
 34. The process of claim 28 , wherein saidisolated plasmid DNA is substantially free of endotoxins and host cellchromosomal DNA.
 35. The process of claim 28 , wherein said plasmid DNAis not exposed to acidic pH or elevated temperatures.
 36. The process ofclaim 28 , wherein said isolated plasmid DNA is produced in a yield ofat least 60%.
 37. The process of claim 28 , wherein said hydrophobicinteraction chromatography is performed in an aqueous solutioncontaining a high concentration of salt.
 38. The process of claim 37 ,wherein said salt is ammonium sulfate.
 39. The process of claim 28 ,wherein said cells are recombinant E. coli cells.
 40. In a process forisolating plasmid DNA from a lysate of a cell containing said plasmidDNA, wherein the improvement comprises using hydrophobic interactionchromatography to purify said lysate, thereby providing isolated plasmidDNA.
 41. The process of claim 42 , wherein said hydrophobic interactionchromatography is performed with at least 1.6M ammonium sulfate.
 42. Theprocess of claim 42 , wherein said hydrophobic interactionchromatography is performed with an Octyl Sepharose 4 FF resin.
 43. Theprocess of claim 42 , wherein said hydrophobic interactionchromatography is performed in an aqueous solution containing a highconcentration of salt.
 44. The process of claim 42 , wherein said saltis ammonium sulfate.
 45. A device for isolating plasmid DNA from cellscontaining said plasmid DNA, comprising: (a) a means for providing fastcell resuspension in a semi-continuous mode; (b) a means for providingmixing and cell lysis in a continuous flow mode; and (c) a means forproviding chilling and mixing to denature and precipitate chromosomalDNA, protein, and RNA.
 46. The device of claim 47 , wherein said meansfor providing mixing and cell lysis in a continuous flow mode comprisesan impeller mixer.
 47. The device of claim 47 , wherein said means forproviding mixing and cell lysis in a continuous flow mode comprises anin-line static mixer.
 48. The device of claim 47 , wherein said meansfor providing mixing and cell lysis in a continuous flow mode comprisesa lysis coil.
 49. The device of claim 47 , further comprising a meansfor performing hydrophobic interaction chromatography.
 50. The device ofclaim 47 , wherein said means for providing chilling and mixing todenature and precipitate chromosomal DNA, protein, and RNA comprises achilled jacketed tank.
 51. A process for isolating plasmid DNA,comprising the steps of: (1) fermenting cells containing said plasmidDNA, harvesting said cells, and washing said cells; (2) exposing saidcells to an alkaline lysis and neutralization agent, thereby forming alysate; (3) performing centrifugation and filtration on said lysate; (4)treating said lysate with RNase at about 37 degrees Celsius for aboutone hour; (5) filtrating said lysate and diluting said lysate with 2volumes of WFI. (6) passing said lysate through a Q Sepharose HP resin,a DEAE 650-S resin, and a Phenyl 650-S resin; and (7) filtrating theeluate from step 6 to yield the final product of isolated plasmid DNA.52. A process for isolating plasmid DNA, comprising the steps of: (a)fermenting cells containing said plasmid DNA, harvesting said cells, andwashing said cells; (b) exposing said cells to an alkaline lysis andneutralization agent, thereby forming a lysate; (c) performingcentrifugation or filtration on said lysate and performing a 1.5 volumedilution with WFI on said lysate; (d) exposing said lysate to an anionicchange resin; (e) washing the nicked and/or relaxed circular plasmid, aswell as residual RNA, off of the resin with about 0.6M NaCl; (f) elutingthe plasmid DNA off of the resin with about 1.9M ammonium sulfite; (g)passing the eluate through a hydrophobic interaction chromatographyresin; and (h) filtrating the eluate to yield a final product ofisolated plasmid DNA.